Structure and Dynamics of an Apoptotic Model Membrane

Abstract

In order to address the dynamic structural changes of cell membranes during apoptosis, we have studied the effect of enzymatically generated ceramide (Cer), in equimolar mixtures of palmitoyl-oleoyl-phosphatidylcholine and egg sphingomyelin (SM). Hydrolysis of SM to Cer was achieved using the well characterized neutral sphingomyelinase from bacillus cereus. By combining high performance thin layer chromatography, synchrotron time-resolved small- and wide-angle x-ray-scattering and photon correlation spectroscopy we were able to correlate the compositional changes of the bilayers to membrane structural adaptations and modifications on the macroscopic level. We found that the hyperbolic increase of Cer levels leads to an instantaneous generation of a gel phase domain. The gel phase forms initially only in the outer membrane leaflet and explains the membrane budding observed previously (1). After about 150 min a constant Cer level of 32 mol % was reached. The membranes, however, continued to swell indicating structural rearrangements due to diffusion processes, vesicle rupture/fusion, or enzyme enclosure. We observe a monotonic growth of vesicle size initiating at about the same time in agreement with vesicle aggregation, reported previously (2). This effect can be understood qualitatively in terms of reduced membrane undulations of the gel phase bilayers. Hence, we present for the first time a structural time-line that bridges the molecular to macroscopic changes occurring during apoptosis. The biological relevance of our results are supported by a remarkable agreement with the kinetics observed in Jurkat cells (3).1. Holopainen, J. M., et al. (2000) Biophys. J. 78, 830-838.2. Ruiz-Arguello, M. B., et al. (1996) J. Biol. Chem. 271, 26616-26621.3. Tepper, A. D., et al. (2000) J. Cell Biol. 150, 155-164.